The cytokinetic ring is a fundamental structure which causes the separation of cells at the end of mitosis. Cytokinetic ring dysfunction is a typical target for antitumor agents.
Conserved in prokaryotic and eukaryotic cells, rings are a plane structure of about 200 nm thickness. They are generally observed by fluorescence microscopy, in a plane perpendicular to their closure (Straight et al., 2003; Sun and Margolin, 1998; Wu et al., 2003). This leads to poor spatial and temporal resolutions.
FIG. 1 illustrates the limitations in the approaches used so far. In a single image, the cytokinetic ring appears as two points, because the image results from the intersection between the focal plane and the actual ring. As a result, broken parts of the ring are not visible, and this can lead to misinterpretation in the effect of drugs. One could argue that the entire ring can still be obtained by scanning the ring along the z direction with n images every 400 nm (deconvolution or confocal microscopy). However, this way of acquiring is not efficient: i/photobleaching due to overexposure of the ring damages the signal and the cell viability; ii/with this method, the acquisition of a single ring requires n times longer than a single plane acquisition, thus reducing the time resolution.
Accordingly, due to the interest of cytokinetic ring, there is a strong need to provide devices and methods allowing to overcome these drawbacks.
Arrays of microwells suitable for cell culture of individual cells have already been disclosed. However, none of them is appropriate to overcome the above-detailed drawbacks for the observation of the cytokinetic ring. As a non-exhaustive illustration, the following articles can be cited.
Ostuni et al (2001, Langmuir, 17, 2828-2834) discloses arrays of microwells, each well being suitable for attaching one cell. The wells have a diameter of less than 100 μm (e.g., 50 and 25 μm) and a depth of less than 50 μm (e.g., 1.3, 5 or 50 μm). The surface within the wells is coated by fibronectin.
Yamamura et al (2005, Analytical Chemistry, 77, 8050-8056) discloses a single-cell microarray and its use for screening. The microchambers are a cylinder having 10-μm width, 12-μm depth and 30-μm pitch.
Ochsner et al (2007, Lab on a Chip, 7, 1074-1077) discloses a device for 3D shape control of single cells. The microwells are coated with fibronectin. The wells' depth is 10 μm and the lateral dimensions were from 81 μm2 to 900 μm2. The 3D shape is controlled by the form of the wells (square, circle, triangles, rectangles, spindles).
Mi et al (2006, Polymer, 47, 5124-5130) discloses a new method of microfabrication of microwells. The microwells can be adapted to contain a single cell.
However, the devices disclosed in these articles imply optical resolution which could be significantly improved, allowing thereby optimal observation quality of key cellular organelles. In addition, none of these documents comprises any teaching about the cytokinetic ring, in particular the orientation of the closure plane thereof.